The Role of Gastrokine 1 in Gastric Cancer

J Gastric Cancer 2014;14(3):147-155  http://dx.doi.org/10.5230/jgc.2014.14.3.147 Review Article

Jung Hwan Yoon, Won Suk Choi, Olga Kim, and Won Sang Park Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul, Korea

Homeostatic imbalance between cell proliferation and death in gastric mucosal epithelia may lead to gastritis and gastric cancer. Despite abundant gastrokine 1 (GKN1) expression in the normal stomach, the loss of GKN1 expression is frequently detected in gastric mucosa infected with Helicobacter pylori, as well as in intestinal metaplasia and gastric cancer tissues, suggesting that GKN1 plays an important role in gastric mucosal defense, and the gene functions as a gastric tumor suppressor. In the stomach, GKN1 is involved in gastric mucosal inflammation by regulating cytokine production, the nuclear factor-κB signaling pathway, and cyclooxygenase-2 expression. GKN1 also inhibits the carcinogenic potential of H. pylori protein CagA by binding to it, and up-regulates antioxidant enzymes. In addition, GKN1 reduces cell viability, proliferation, and colony formation by inhibiting cell cycle progression and epigenetic modification by down-regulating the expression levels of DNMT1 and EZH2, and DNMT1 activity, and inducing apoptosis through the death receptor- dependent pathway. Furthermore, GKN1 also inhibits gastric cancer cell invasion and metastasis via coordinated regulation of epithelial mesenchymal transition-related protein expression, reactive oxygen species production, and PI3K/Akt signaling pathway activation. Although the modes of action of GKN1 have not been clearly described, recent limited evidence suggests that GKN1 acts as a gastric- specific tumor suppressor. This review aims to discuss, comment, and summarize the recent progress in the understanding of the role of GKN1 in gastric cancer development and progression.

Key Words: GKN1 protein; Homeostasis; Gastritis; Stomach neoplasms; Tumor suppressor gene

Introduction ingested noxious agents, and acidic pH, may lead to diseases such as gastritis and gastric cancer. Therefore, protective and reparative Generally, the gastrointestinal epithelium is characterized by mechanisms are essential to rapidly restore gastric mucosal integrity a very high cellular turnover rate, which leads to renewal of the by stimulating migration of epithelial cells over denuded areas (res- epithelium every 3 to 5 days.1 Continuous processes of cell prolif- titution), increasing mucus production, and reestablishing epithelial eration, differentiation, and self-renewal are counterbalanced by proliferation and differentiation programs.2 Although endogenous senescence and/or apoptosis in normal gastric mucosa. To maintain molecules, such as members of the trefoil factor 1 protein family, homeostasis, a critical balance between cell proliferation and death protect the gastric mucosa from noxious environments,3 many of is required, and complex signaling pathways and transcriptional their underlying molecular mechanisms have not been clearly elu- regulators control it. Otherwise, homeostatic imbalance, caused cidated. by several factors, including infection with Helicobacter pylori, Gastrokine 1 (GKN1), also called antral mucosal protein (AMP)-18, is a protein synthesized by the cells of the antral gastric Correspondence to: Won Sang Park mucosa and shows growth factor or ‘cytokine-like’ activity toward Department of Pathology, College of Medicine, The Catholic University 4 of Korea, 222 Banpo-daero, Seocho-gu, Seoul 137-701, Korea gastric epithelial cells. Immunoelectron microscopy indicated that Tel: +82-2-590-1192, Fax: +82-2-537-6586 the GKN1 protein is localized within the granules just under the E-mail: [email protected] apical plasma membrane, suggesting that it is a secreted rather Received August 12, 2014 4 Revised September 12, 2014 than an integral membrane protein. Early work reported that Accepted September 13, 2014

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GKN1 protects the antral gastric mucosa and promotes healing by hydrophobic region and BRICHOS domain of GKN1, but not the facilitating restitution and proliferation following injury.4 Interest- COOH-terminal region, significantly reduce cell viability, prolif- ingly, this protein is abundantly expressed in the gastric mucosa of eration, and colony formation of AGS gastric cancer cells by regu- healthy individuals, but is down-regulated or absent in gastric can- lating cell cycle progression, and minimize epigenetic modification cer tissues.4 Because a disruption in gastric homeostasis may result by inhibiting the expression of DNMT1 and EZH2, and DNMT1 in the transformation of normal epithelial cells into cancer cells and activity. Therefore, these findings indicate that the NH2-terminal permit cancer cells to proliferate and invade, there is no doubt that hydrophobic region and BRICHOS domain of GKN1 might be the inactivation of GKN1 may render the gastric mucosa vulnerable to main functional domain for tumor suppressor activity.12 carcinogens or gastric injury, and eventually trigger genetic altera- Two isoforms of the GKN1 protein with different NH2- tions in cancer-related genes, including oncogenes and tumor sup- terminal residues, aspartic acid (D) and asparagine (N), were found pressor genes. in gastric endoscopy biopsy samples, and the isoform containing Although limited published evidence suggests that GKN1 may asparagine was decreased or absent in some H. pylori-positive play an important role in gastric epithelial homeostasis and gastric patients.13 Therefore, these two isoforms might play different roles carcinogenesis, its definitive biological functions remain unclear. In in H. pylori-infected gastric mucosa. Previously, we reported that this review, we discuss the current understanding of the biological an aspartic acid or asparagine variant at codon 13 in the NH2- roles of the GKN1 tumor suppressor in the development and pro- terminal region, but not alanine variant of the pGKN1D13A mutant, gression of gastric cancer. was found to be important in the anti-growth function of GKN1.12 Therefore, it seems that genetic alterations or loss of expression

Characteristics of Gastrokine 1 of the NH2-terminal hydrophobic region and BRICHOS domain of GKN1 might lead to the functional inactivation of the GKN1 The protein, previously known as AMP-18, CA11, FOVEO- protein. However, to explain how GKN1 expression is controlled LIN, and TFIZ,5-8 was formally named ‘GKN1’ by the HUGO in normal gastric mucosa, further studies are important to delineate Gene Nomenclature Committee (HGNC no. 23217) for its gastric- the molecular mechanisms regulating transcription and biological specific expression and its highly conserved presence in the gastric roles of GKN1 in gastric mucosal epithelia. mucosa of many mammalian species.5,6 Three members of the GKN family, GKN1, GKN2, and GKN3, are located on chro- Gastrokine 1 in Non-Neoplastic mosome 2p13.3 and arranged within a 60-kb genomic interval.9 Gastric Mucosa The structures of the GKN genes exhibit nearly identical genomic architecture, with each containing six exons separated by relatively GKN1 is a unique gastric-specific protein, whose expres- short introns.9 GKN genes encode small (from 181- to 184-amino sion is confined to the gastric epithelium, except for trace levels acid) proteins; and contain the enigmatic BRICHOS domain and in the uterus and placenta.5 GKN1 protein and mRNA are local- a COOH-terminal segment showing considerable divergence be- ized in surface mucous cells of the gastric antrum and fundus in 6,13-16 tween the GKN paralogs and the hydrophobic NH2-terminal sig- humans. GKN1 is involved in filling the lumen of the surface nal peptide, the processing of which is predicted to generate about layer of epithelial cells to maintain mucosal integrity and regulate 160 mature amino acid proteins with a molecular mass of approxi- cell proliferation and differentiation.5,6 In addition, GKN1 is present 9,10 mately 18 kDa. Of these, the NH2-terminal hydrophobic region in the secretion granules of gastric mucosal epithelial cells during acts as a transmembrane anchor and/or signal peptide.10 Clinically, immunogold-labeling experiments4 and, therefore, is predicted to the proteins in the BRICHOS superfamily are associated with de- function as a secreted protein acting primarily in the extracellular mentia, respiratory distress syndrome, and cancer.10,11 Molecular and luminal environment.9 As mentioned earlier, the gastrointes- studies on the BRICHOS domain function have suggested that it tinal epithelium is characterized by a very high cellular turnover has a range of possible roles, including intracellular trafficking, pro- rate, which leads to renewal of the epithelium every 3 to 5 days.1 peptide processing, chaperone function, and secretion.11 However, GKN1 also facilitates regeneration of the injured colonic epithelium the biological activities of the BRICHOS domain in GKN1 have by accelerating migration of surviving cells at the edges of wounds, not been elucidated. We previously reported that the NH2-terminal and also stimulating cell proliferation to resurface the injured mu- 149 The Role of Gastrokine 1 in Gastric Cancer cosa after cell detachment, apoptosis, and necrosis.4,5 However, by chronic H. pylori infection. A critical balance between pro- and subsequent studies have reported that overexpression of GKN1 anti-inflammatory signals maintains a healthy gastric homeostasis, inhibits cell proliferation and induces Fas-mediated apoptosis and which effectively neutralizes harmful environmental insults to pre- senescence through p16/Rb pathway activation in gastric epithelial vent excessive tissue damage.20 Therefore, imbalance between pro- cells.16-19 Although the molecular mechanisms that explain these and anti-inflammatory cytokines may render the gastric mucosa opposite effects of GKN1 in colon and gastric epithelial cells re- vulnerable to injury, resulting in gastritis. Among inflammatory main unclear, it is possible that GKN1 might inhibit or stimulate mediators, nuclear factor (NF)-κB is a potent transcriptional factor cell growth in a cell type-specific and context-dependent manner. that orchestrates many biological functions essential for inflam- Therefore, more work is needed to better understand the bioactivity matory and immune processes induced by H. pylori, and whose of GKN1 in maintaining the homeostasis of normal gastric mu- activation stimulates interleukin-1 (IL-1) and tumor necrosis fac- cosa. tor-α production.21,22 In addition to the role of cytokine production, Generally, gastric mucosal inflammation is believed to be caused aberrant activation of NF-κB leads to cell growth and resistance

Fig. 1. Role of GKN1 in gastric cancer. GKN1 induces apoptosis through the death receptor dependent pathway and inhibits Helicobacter pylori CagA injection into gastric epithelial cells. GKN1 also reduces reactive oxygen species production by up-regulating the expression of antioxidant enzymes, such as MnSOD and catalase. At the intracellular level of gastric epithelial cells, GKN1 induces miR-185 expression by down-regulating c- myc, which in turn inhibits epigenetic modification of genomic DNA and stimulates the expression of negative cell cycle regulators, including p53, p21, and p16. In addition, GKN1 inhibits epithelial mesenchymal transition by inactivating PI3K/Akt and β-catenin signaling pathway, resulting in the disruption of invasion and metastasis of gastric cancer cells. Blue line: stimulator, Red line: inhibitor. GKN1 = gastrokine 1; ROS = reactive oxygen species; NF-κB = nuclear factor-κB; COX2 = cyclooxygenase-2; CASP3 = caspase 3; CASP8 = caspase 8; CDH1 = cadherin 1; DNMT1 = DNA methyltransferase 1; EZH2 = enhancer of zeste homolog 2; PI3K = phosphatidylinositol 3-kinase; IKKα/β = IκB kinase α/β. 150 Yoon JH, et al. to apoptosis in gastric cancer cells.23,24 Interestingly, ectopic GKN1 biomarker for evaluating the severity of gastritis and CagA-positive expression was found to suppress the activation and nuclear trans- H. pylori infection. location of NF-κB by inhibiting degradation and phosphorylation GKN2, also known as TFIZ1 and Blottin, is expressed in sur- of NF-κB specific inhibitor (IκB) and inactivating IKKα/β in AGS face pit cells in both the corpus and antrum, and are thought to be gastric cancer cells (Fig. 1).25 secreted.8,39-41 GKN2 gene expression was shown to be significantly Furthermore, GKN1 induces a significant increase in the ex- reduced in AGS and KATO-III gastric adenocarcinoma cell lines pression of IL-17, but markedly decreases the expression of IL- by the p50 and p65 subunits of NF-κB, and co-transfection with 6, IL-10, and cyclooxygenase-2 (COX-2), which are correlated IκB resulted in up-regulation of the GKN2 gene.2 Interestingly, with tumor progression and serve as independent predictors of GKN2 alone did not affect cell viability, whereas co-transfection poor survival in gastric cancer (Fig. 1).25-28 The IL-17 family of with GKN1 and GKN2 in vitro showed that GKN2 inhibited the cytokines performs numerous immune regulatory functions re- effect of GKN1 on cell viability, proliferation, and apoptosis by lated to local tissue inflammation by inducing and mediating pro- down-regulating miR-185 expression and inducing epigenetic inflammatory responses.29 IL-17 acts upon gastric epithelial cells modification. Furthermore, the expression of GKN2 was regulated to release IL-8, which recruits neutrophils,30 and is associated with in a GKN1-dependent manner by inactivating the NF-κB signal- prolonged survival in gastric cancer patients.31 In contrast, IL-10 is ing pathway.42 Therefore, GKN2 might play an important role in an anti-inflammatory cytokine that suppresses the production of gastric mucosal homeostasis by regulating GKN1 activities. These pro-inflammatory cytokines through the inhibition of Th1 lym- data agree with the previous findings that GKN2 did not induce phocytes and stimulation of B and Th2 lymphocytes, in order to any significant effects in the cell wounding assay in HGT-101 gas- down-regulate the inflammatory response.32 In particular, IL-6 tric cancer cells.40 However, GKN2 expression was also reduced in H. is generally considered to be a pro-inflammatory and pleiotropic pylori-infected gastritis37 and gastric cancers.43,44 Moreover, GKN2 cytokine involved in tumor initiation, promotion, and progression.33 decreased cell growth in SGC-7901 gastric cancer cells in vitro.43 A COX-2 expression in the injured mucosa plays a significant role recent study confirmed that GKN2 inhibits proliferation, migration, in the repair processes and defense mechanisms of the stomach.34 and invasion of gastric cancer cells, and arrests the cell cycle at the COX-2 inhibition has been shown to suppress cell proliferation G1/S transition phase,44 prompting recognition of GKN2 as a puta- and induce apoptosis in many gastrointestinal cancer cell lines.35 tive stomach-specific tumor suppressor gene and implying its po- Therefore, these findings imply that GKN1 may play an important tential use in gastric cancer therapy. While the basis of these con- role in gastric mucosal inflammation by mediating cytokine pro- troversial results is unclear, we speculate that GKN2 might suppress duction and suppression of NF-κB and COX-2, thereby contribut- gastric cancer cell growth, but inhibit GKN1-induced cell death to ing to the maintenance of gastric mucosal homeostasis. maintain homeostasis in normal gastric mucosa. Further efforts to It has been reported that H. pylori infection and long-term clarify the molecular mechanisms underlying GKN2 functions will non-steroidal anti-inflammatory drug administration down-reg- provide more comprehensive information about the use of GKN2 ulate the expression of GKN1 in gastric mucosal epithelial cells.15,36 in anticancer or mucosal protective therapeutics. In accordance with these observations, the GKN1 mRNA transcript was strongly up-regulated in the gastric transcriptome after Gastrokine 1 in the Development of H. pylori eradication and cessation of gastritis.37 Previously, we also Gastric Cancer found that the presence of H. pylori CagA was inversely correlated with GKN1 expression in non-neoplastic gastric mucosa.38 Fur- GKN1 mRNA is one of the most abundant transcripts in the thermore, the expression of GKN1 was significantly reduced in the normal gastric mucosa, accounting for approximately 1% of total cases with H. pylori CagA positivity, mononuclear cell infiltration, gastric mRNA.45 Interestingly, it was demonstrated that individuals atrophy, and intestinal metaplasia. In addition, GKN1 predicted with a lower expression of GKN1 have an increased risk of devel- the risk of atrophy and intestinal metaplasia with an area under oping gastric disease.14 Numerous studies have described frequent the receiver operating characteristic curve value of 0.865 and 0.973, reduction or complete loss of GKN1 expression in gastric tumors, respectively.38 Taken together, these findings provide evidence that including adenomas and adenocarcinomas.6,7,15,16 Loss of GKN1 ex- the expression levels of GKN1 might be a predictive and diagnostic pression in gastric cancer was first described in differential display 151 The Role of Gastrokine 1 in Gastric Cancer analysis.46 In immunohistochemical analysis, loss of GKN1 expres- into cells by binding to CagA at the extracellular level,51 suggesting sion was detected in 36 (90%) and 170 (89.5%) of gastric adenomas that GKN1 may counteract the CagA-induced genetic alterations. and carcinomas, respectively.16 Statistically, the loss of GKN1 ex- These results were corroborated by those of Moss and colleagues,14 pression in gastric cancer showed no association with tumor dif- who reported that individuals with a lower expression of GKN1 ferentiation, depth of invasion, location, and lymph node metastasis, have an increased risk of developing gastric cancer. Therefore, it is or clinical stage.7,16 Therefore, GKN1 inactivation may commonly expected that the presence of GKN1 helps to maintain the homeo- occur as an early event in the development of gastric tumorigen- stasis of normal gastric mucosa, even if H. pylori infection exists. esis. However, Moss et al.14 revealed the loss of GKN1 expression In contrast, inactivation of the GKN1 gene may lead to a defect in 78% of diffuse-type and 42% of intestinal-type gastric cancers, in the gastric mucosal barrier, rendering gastric mucosa subject to and this loss of expression was positively correlated with that of exposure to carcinogens, including H. pylori CagA, and eventually GKN2. Additionally, the authors found that the combined loss of evoke subsequent genetic alterations of tumor suppressor genes or GKN1 and GKN2 expression may correlate with shorter overall oncogenes involved in gastric carcinogenesis. survival in the intestinal-type gastric cancer. Thus, further clinical validation studies in larger populations are necessary to validate the Gastrokine 1 in the Progression of applicability of GKN1 and GKN2 as prognostic and/or differentia- Gastric Cancer tion markers for gastric cancer patients. Although it is well known that GKN1 expression is undetected Epithelial mesenchymal transition (EMT) is a normal physi- in gastric tumors, the molecular mechanisms underlying GKN1 ologic process, during which cells lose epithelial characteristics and inactivation remain unclear. Chromosomal alterations, including gain more motile mesenchymal features allowing relocation of the deletions or loss of heterozygosity at 2p13.3, where GKN genes are cells to new sites and participation in tissue and organ formation.52 located, have not been found in gastric tumors.47-49 Previously, we This phenomenon is observed in response to injury, organ fibro- reported that there was no somatic mutation in the coding regions sis, and cancer. In cancers originated from epithelial cells, EMT of GKN1 and the hypermethylation of the GKN1 promoter was converts epithelial cells into cells with invasive characteristics that observed in 2 (8.0%) of 25 gastric cancers. However, significant enable them to destroy and move into surrounding normal tissues, loss of the GKN1 gene at DNA and mRNA levels was detected in including blood and lymphatic vessels, and to spread systemically gastric cancer tissues. Thus, we conclude that DNA and mRNA into other tissues or organs. In addition, circulating tumor cells are transcript loss of the GKN1 gene, but not genetic and epigenetic thought to undergo EMT, whereby epithelial traits are lost in favor alterations, may play a major role in the loss of GKN1 expression of more mesenchymal traits, thus acquiring invasive capabilities and in gastric cancer.16 plasticity.53 Unfortunately, most gastric cancer patients are diag- Chronic infection with H. pylori perpetuates chronic gastritis, nosed at the advanced stages of the disease, which contribute to the which progresses to mucosal atrophy, intestinal metaplasia, dys- high lethality rate in the world.54 The identification of signals that plasia, and carcinoma.50 As described above, GKN1 expression is lead to EMT and cancer cell migration remains a central challenge down-regulated in H. pylori-infected gastric mucosal tissue.13,15,36,37 in cancer research, and a better understanding of the molecu- These observations invoke the hypothesis that H. pylori infection lar mechanisms of invasion and metastasis of gastric cancer may might regulate the expression of GKN1 protein. In recognition provide new insight and information to develop novel therapeutic of this possibility, we analyzed the effects of H. pylori CagA on targets for treatment of gastric cancer patients. GKN1 expression in gastric epithelial cells. Interestingly, H. pylori As described above, turning epithelial cancer cells into mesen- CagA-induced reactive oxygen species (ROS) reduced the expres- chymal cells was proposed to be the critical process for the acqui- sion of the GKN1 protein by decreasing GKN1 DNA and mRNA sition of the invasive and metastatic phenotype of epithelial cancer copy numbers in gastric cell lines and mucosal tissues of humans cells during tumor progression. Previously, it was reported that and mice.51 As shown in Fig. 1, the ectopic expression of GKN1 ROS stimulated EMT,55 and the activation of PI3K/Akt signal- inhibited CagA-induced ROS production and GKN1 copy num- ing was detected in cells undergoing EMT.56 Our subsequent study ber change via overexpression of antioxidant enzymes, MnSOD, found that GKN1 induced the conversion of spindle-shaped cells and catalase. In addition, GKN1 prevented the injection of CagA with abundant cytoplasms to circular-shaped cells, and suppressed 152 Yoon JH, et al. cell migration and invasiveness in wound healing, transwell che- DNMT1 and EZH2 expression in gastric cancer tissues and various motaxis, and invasion assays.57 In addition, GKN1 significantly re- gastric cancer cell lines. Furthermore, RNA interference-mediated duced ROS levels by up-regulating antioxidant enzymes, including knockdown of GKN1 in HFE-145 non-neoplastic gastric epithe- MnSOD and catalase, and completely abrogated expression of the lial cells, which express GKN1 protein, induced cell growth, while PI3K/Akt pathway proteins, concomitant with the re-expression GKN1 was sufficient to induce cell cycle arrest in AGS, MKN1, of E-cadherin and decreased expression of fibronectin, vimentin, and MKN28 gastric cancer cells. Moreover, GKN1-induced miR- nuclear β-catenin, slug, snail, and cyclin D1 (Fig. 1).57 This limited 185 converted hypermethylated CDKN2A and E-cadherin to the evidence indicates that GKN1 might inhibit the invasion and me- unmethylated forms in AGS cells (Fig. 1).17 Therefore, these results tastasis of gastric cancer cells via coordinate regulation of expres- suggest that GKN1 may inhibit gastric carcinogenesis through the sion of EMT-related proteins, ROS production, and the activation induction of miR-185, thereby modulating epigenetic alterations in of the PI3K/Akt signaling pathway. Based on the above data, we cell cycle regulatory components. conclude that GKN1 may inhibit not only the development, but Regarding cell death, ectopic expression of GKN1 in AGS gas- also the progression of gastric cancer. tric cancer cells induced activation of the apoptosis-related proteins, including cleaved caspase-8, caspase-3, and poly-(ADP-ribose) Biological Activities of Gastrokine 1 polymerase, whereas no change in expression of the mitochondrial pathway-related proteins was observed (Fig. 1).16 Thus, we conclude Numerous studies have described frequent loss of GKN1 ex- that GKN1 may induce apoptosis via the death receptor-mediated, pression in gastric cancer and its anti-proliferative activity in gastric but not the mitochondria-mediated pathway. Similarly, Rippa et epithelial cells, suggesting that GKN1 may be a gastric specific al.19 reported that AGS cells overexpressing GKN1 showed higher tumor suppressor. However, the definitive biological functions expression of Fas receptor and sensitivity to Fas-ligand-induced of GKN1 have not yet been identified. In colonic epithelial cells, apoptosis. In addition, Chen et al.62 identified the cholecystokinin- GKN1 exerted its protective effects by increasing accumulation B/gastrin receptor (CCKBR) as a putative receptor for GKN1 of specific tight and adherence junction proteins, and protecting protein by affinity purification and mass spectrometry analysis. The their loss after injury.58 Toback et al.4 also proposed that the GKN1 authors found that recombinant human AMP-18 (GKN1) bound protein could have some mitogenic impacts on intestinal epithelial to the plasma membrane of keratinocytes in normal human oral cells. In contradiction to its protective and mitogenic activity, Shio- mucosal tissue, and that CCKBR was co-immunoprecipitated with zaki et al.7 found that GKN1 was capable of inhibiting the pro- exogenous AMP-18 in diverse epithelial cells. In gastric epithelial liferation of MKN28 cells after transfection. Similarly, subsequent cells, binding of gastrin to CCKBR induced the expression and re- studies demonstrated that GKN1 not only inhibited cell prolifera- lease of heparin-biding epidermal-like growth factor, which sub- tion, but also induced apoptosis and senescence.16-19,59,60 As ex- sequently transactivated the epithermal growth factor receptor and pected, restoration of GKN1 expression resulted in cell cycle arrest downstream signaling pathways.63 However, no data has been re- at the G1/S or G2/M phases by down-regulating positive cell cycle ported concerning the existence of a receptor for secretary GKN1 regulators, including CDK4, cyclin D1, E2F, cdc25, and cyclin B, at the membrane of gastric mucosal epithelial cells, to which GKN1 and up-regulating negative cell cycle regulators, including p16 and may directly bind to. Thus, to investigate whether GKN1 directly p21 (Fig. 1).17,18,61 Using proteomics analysis, Yan et al.61 identified binds to CCKBR at the membrane of gastric epithelial cells, and if the pro-oncogenic protein enolase-α (ENO1), which prevented so, how GKN1 regulates the complex of gastrin and CCKBR and GKN1-dependent growth inhibition and cell cycle arrest and, thus, maintains the gastric mucosal homeostasis will be of great interest might be a possible target and antagonist of GKN1 activity in gas- to study further. tric cancer cells. Recently, we examined miRNA-185 to elucidate the underlying molecular mechanisms of GKN1-dependent growth Conclusion inhibition. We demonstrated that GKN1 suppressed gastric cancer cell growth by inducing endogenous miR-185 that directly targeted Recent evidence has demonstrated that GKN1 may play an epigenetic effectors, such as DNMT1, EZH2, and HDAC (Fig. important role in the maintenance of gastric mucosal homeostasis 1).17 In addition, GKN1 expression was inversely correlated with and function as a gastric specific tumor suppressor. Although the 153 The Role of Gastrokine 1 in Gastric Cancer molecular mechanisms underlying its tumor suppressor activity are 5. Martin TE, Powell CT, Wang Z, Bhattacharyya S, Walsh-Reitz largely unknown, it was found that GKN1 is involved in gastric MM, Agarwal K, et al. A novel mitogenic protein that is highly mucosal inflammation by regulating production of inflammatory expressed in cells of the gastric antrum mucosa. Am J Physiol mediators, including NF-κB, COX-2, and cytokines. In addi- Gastrointest Liver Physiol 2003;28:G332-G343. tion, GKN1 inhibits the carcinogenic potentials of H. pylori CagA 6. Oien KA, McGregor F, Butler S, Ferrier RK, Downie I, Bryce through the direct binding to CagA at the extracellular level, and S, et al. Gastrokine 1 is abundantly and specifically expressed increases the expression of antioxidant enzymes at the intracellular in superficial gastric epithelium, down-regulated in gastric level. Furthermore, GKN1 interferes with cell growth and invasion carcinoma, and shows high evolutionary conservation. J Pathol by suppressing cell cycle progression and EMT, and induces apop- 2004;203:789-797. tosis via the death receptor-dependent pathway, thereby inhibiting 7. Shiozaki K, Nakamori S, Tsujie M, Okami J, Yamamoto H, Na- not only the development, but also the progression of gastric cancer. gano H, et al. Human stomach-specific gene, CA11, is down- Thus, it is plausible that modulating GKN1 activity and stimulating regulated in gastric cancer. Int J Oncol 2001;19:701-707. its anti-cancerous effects could significantly influence the develop- 8. Westley BR, Griffin SM, May FE. Interaction between TFF1, a ment of novel cancer therapeutics, which would ultimately achieve gastric tumor suppressor trefoil protein, and TFIZ1, a brichos the goal of gastric cancer prevention and remission. Future stud- domain-containing protein with homology to SP-C. Biochem- ies to clarify the biological activity and regulatory mechanisms of istry 2005;44:7967-7975. GKN1 will be pivotal to develop GKN1 as a potential therapeutic 9. Menheniott TR, Kurklu B, Giraud AS. Gastrokines: stomach- target for gastric cancer patients. specific proteins with putative homeostatic and tumor suppressor roles. Am J Physiol Gastrointest Liver Physiol Acknowledgments 2013;304:G109-G121. 10. Hedlund J, Johansson J, Persson B. BRICHOS - a superfam- This work was supported by the Basic Science Research Pro- ily of multidomain proteins with diverse functions. BMC Res gram through the National Research Foundation of Korea (NRF) Notes 2009;2:180. funded by the Ministry of Education, Science and Technology 11. Sánchez-Pulido L, Devos D, Valencia A. BRICHOS: a con- (2012R1A2A2A01002531). served domain in proteins associated with dementia, respiratory distress and cancer. Trends Biochem Sci 2002;27:329-332. References 12. Yoon JH, Choi YJ, Choi WS, Nam SW, Lee JY, Park WS. Func- tional analysis of the NH2-terminal hydrophobic region and 1. Hall PA, Coates PJ, Ansari B, Hopwood D. Regulation of cell BRICHOS domain of GKN1. Biochem Biophys Res Commun number in the mammalian gastrointestinal tract: the impor- 2013;440:689-695. tance of apoptosis. J Cell Sci 1994;107:3569-3577. 13. Nardone G, Rippa E, Martin G, Rocco A, Siciliano RA, Fiengo 2. Baus-Loncar M, Lubka M, Pusch CM, Otto WR, Poulsom R, A, et al. 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